Polymalic Acid-Based Multi-Functional Drug Delivery System

ABSTRACT

A structured drug system that is useful for delivering a drug payload to a specific tissue or cell type is disclosed. The system is based on purified polymalic acid. This polymer isolated from natural sources is biocompatible, biodegradable and of very low toxicity. The polymer is extremely water soluble and contains a large number of free carboxyl groups which can used to attach a number of different active molecules. In the examples disclosed N-hydroxysuccinimide esters of the carboxyl groups are used to attach such molecules. The active molecules include monoclonal antibodies to promote specific cellular uptake and specific pro-drugs such as antisense nucleic acids designed to modify the cellular metabolism of a target cell. The pro-drugs are advantageously linked by a somewhat labile bond so that they will be released under specific conditions. In addition, the system contains amide-linked valine to encourage membrane disruption under lysosomal conditions. Polyethylene glycol groups are attached to extend the drug system&#39;s circulation half-life. In addition, fluorescent reported groups can be readily included to aid in visualizing and confirming drug system targeting. The drug system can deliver treatments for a wide range of diseases and is specially advantageous for treatment of neoplasms.

The present application is based on U.S. Provisional Patent ApplicationSer. No. 60/527,330 filed 5 Dec. 2003 and claims priority from thatapplication.

BACKGROUND OF THE INVENTION AND DESCRIPTION OF THE PRIOR ART

The present invention relates to the field of targeted delivery of drugsand more specifically involves a multifunctional targeted drug deliveryvehicle.

Currently several different molecular scaffolds are used in thesynthesis of drug vehicles; notable examples areN-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (20-30 kDa) [42] andother derivatives of polyacrylic acid. However, these are not consideredto be biodegradable [43, and references therein], because of theircarbon-carbon backbone, and they are problematic due to inevitablecontamination by hazardous acrylic acid [44]. Other, degradablescaffolds (e.g. poly(L-glutamic acid) [45] may have unfavorableproperties, like rotational restriction around the peptide bond orlimited solubility in organic solvents desirable for chemical synthesisand product purification, and, in addition, is supportive ofimmunogenicity in the structural proximity to other potentiallyimmunogenic structures due to the high hydrogen bond forming capacity ofthe peptide backbone [45, 46, 47].

Antisense Technology. Antisense oligonucleotides (oligos) that bind andinactivate specific RNA sequences may be one of the best tools forstudying gene function, regulation of gene expression, interactionsbetween gene products, and validation of new therapeutic targets fordrug development. Antisense oligos offer the promise of safe andeffective therapeutics for viral diseases, cancers, and otherdevastating diseases. Specific antisense oligos that mimic DNA templatefor RNA production are used to bind to complementary RNA and to preventprotein translation (e.g., of tumor markers) [1].

There are promising data on the use of antisense technology in gliomas.Glioma growth in vitro and in nude mice can be inhibited by antisense totelomerase [2]. A pilot study showed that antisense to IGF-I receptorinduced glioma cell apoptosis and resulted in clinical improvements ofpatients [3]. Several clinical trials are currently using antisenseoligos for treatment of other cancers [4]. These studies take advantageof new generation antisense oligos free from insufficient specificity,stability, and non-antisense effects [5]. The most promising varietiesof improved oligos are Morpholinos oligos and peptide nucleic acid (PNA)oligos. These varieties have the highest sequence specificity of allantisense types, and maintain this specificity over a very broadconcentration range [6, 7, 8, 9, 10 and 11]. A new, rapidly evolving,variant of antisense approach is represented by small interfering RNAs(siRNAs) that are also highly potent gene expression silencers andpotential anticancer drugs.

Combined blocking of several molecular markers in vitro and in vivo toprevent tumor progression. This approach has long been used successfullyin cancer chemotherapy but has not yet been applied to targetingspecific tumor markers. Only following the development of gene/proteinarray approaches, did it became possible to obtain and correlate data onconcerted changes of specific genes during tumor progression andrecurrence. Such concerted changes offer a possibility of counteractingsimultaneous alterations of several genes in the hope of efficientlyblocking tumor development and progression. There are several candidategenes for blocking to stop glioma growth and spread including tyrosinekinase receptors (e.g., EGFR), some growth factors, and antiapoptoticgenes that can be potentially used in combination with chemotherapeuticagents to more efficiently prevent tumor growth [12, 13, 14, 15, and16]. Our earlier studies identified another potential candidate protein,laminin-8, which is overexpressed in brain and breast tumors, correlateswith poor prognosis of gliomas and is involved in glioma invasion.

Drug delivery. For direct targeting of cancer cells to treat tumors, thedrugs, e.g., monoclonal antibodies, antisense oligos or small molecules(such as Tarceva (erlotinib)), should be able to penetrate the cellmembrane. There are three basic methods for intracellular drug delivery,passive diffusion through aqueous channels or pores in the membrane,passive diffusion of lipid-soluble drugs through dissolution in thelipids of the membrane, and carrier-mediated active transport (viralvectors, liposome-mediated gene transfer system, special chemicals) [16,17]. Brain tissue is especially difficult to treat with drugs because ithas a special blood brain barrier with tight junctions between brainmicrovascular endothelial cells that prevent penetration ofwater-soluble and ionized or polar drugs [18, 19].

High molecular weight molecules have recently received special attentionbecause of the enhanced permeability and retention (EPR) effect observedin cancer tissue for macromolecules and lipids (MW>45 kDa) [20, 21, and22]. Unlike small molecule anticancer drugs used today, which do notdiscriminate tumor from normal tissue, macromolecular (or polymeric)drugs can target tumors with high selectivity through the EPR effect[22, 23]. One such promising drug carriers, poly-L-malic acid (PMLA),has been developed by one of the present inventors [24, 25].

SUMMARY OF THE INVENTION

It is an object of the invention is to solve the problems that haveformerly plagued drug carrier systems by using polymalic acid, whichcarries an abundance of functional carboxylic groups, at least about 50of such groups and about 500 per polymalic acid molecule of mass 50,000.The polymalic acid is readily available in a range of sizes from amolecular mass of 2,500 to at least 100,000. This allows the attachmentof a large number of biologically active functional molecular modules toachieve:

-   -   a high variability in the kind and number of tissue targeting        molecules per drug carrier molecule;    -   a high variability in the kind and number of conjugated drug        modules (pro-drugs) per drug carrier molecule;    -   an ability to control solubility by having groups carrying        either hydrophobic or hydrophilic residues in addition to the        functional modules;    -   an ability to conjugate, other than the described functional        modules, additional groups, such as PEG, which are active in        protection of the carrier system against degradation e.g., to        increase lifetime in the blood circulation system;    -   a carrier scaffold that is biodegradable, along with many other        residues used as building units of the drug delivery system;    -   a system with no use of viral components;    -   a delivery system with functional modules (mABs or other tumor        cell surface receptor ligands), the kind of drug (antisense        oligonucleotides to malignantly expressed genes), and a totally        high molecular mass of the drug delivery system account for high        specificity towards tumor tissue (EPR-effect); and    -   a drug system of low toxicity allowing rat dosages as high as 5        mg/kg rat body weight.

A further object of the invention is:

-   -   to facilitate synthesis of a drug delivery system by avoiding        convoluted synthetic methods and uncontrollable side reactions;    -   to build in favorable solubility and other properties that        provide an easy purification;    -   to achieve maximum yields that allow defined stoichiometry and        the reproducibility of each individual chemical conjugation        reaction;    -   to lay the structural and synthetic fundaments for a high        variability regarding the kind and number of conjugated        functional modules; and    -   to provide a technology that allows simple scale up of the drug        delivery system.

The technical invention includes:

-   -   the choice of drug delivery system employing polymalic acid as a        backbone or scaffold that carries a very high number of reactive        carboxylic groups (about 500 for a scaffold of molecular mass        50,000);    -   activation of most (ideally all) of these carboxylic groups by        forming their NHS-esters;    -   chemically independent preparation of functional modules, which        carry single amino groups as nucleophiles for substitution at        the NHS-activated carboxyl groups;    -   preparing the activated scaffold and each of the reactive        functional modules separately;    -   combining and reacting the NHS-activated scaffold and each        reactive functional module independent but in a well defined        sequential order, allowing purification and verification of the        desired intermediates/products after each addition of a newly        added functional module;    -   combining these reactants in stoichiometric amounts;    -   achieving high and reproducible yields of conjugates; and    -   avoiding side reactions of newly added reactive functional        modules with the already conjugated modules by choosing a well        organized hierarchy of sequential additions for the conjugation        of the next incoming functional modules.

The design and synthesis of a carrier or scaffold is described, whichtransports a covalently conjugated drug to a targeted tissue, binds tocell surface receptors of the tissue, internalizes into endosomes,escapes the endosomes into the cytoplasm, and releases reactive freedrug in the cytoplasm by chemical reaction with glutathion and othersulfhydryl groups of the cytoplasmic content. The specificity of highmolecular mass drug vehicles or even particles rests on the both thetumor tissue targeting by tumor-specific conjugated targeting moleculesand their enhanced permeability and retention in tumors (EPR-effect)that solely originates from their high molecular mass (>20000)[40, 41].

The scaffold poly(malic acid) (PMLAH) used in the present patentapplication contains a main chain ester linkage, is biodegradable [27,and references therein] and of a high molecular flexibility [49],soluble in water (when ionized) and organic solvents (in its acid form),non-toxic, and non-immunogenic [27, and references therein]). Drugcarrying PMLAH has been mainly synthesized by ring-openingpolymerization of derivatized malic acid lactones [27, and referencestherein]. Synthesis of Doxorubicin-poly(malic acid) has been reportedfrom chemically synthesized poly(β-D, L-malic acid) [49]. The synthesisof drug vehicle from naturally occurring PMLAH has not been carried out.The kind of highly functional drug delivery system described in thepresent patent application has not been previously disclosed.

The carrier consists of poly(β-L-malic acid) (PMLA) representing themolecular backbone or scaffold that is chemically conjugated at itscarboxylic groups at defined ratios with a variety of functional modulesthat perform the following tasks: (1) delivery of a pro-drug via areleasable functional module that becomes effective in the cytoplasm,(2) directing the carrier towards a specific tissue by binding to thesurfaces of cells (e.g. a monoclonal antibody (mAB)), (3)internalization into the targeted cell through endosomes (usually viainternalization of a targeted surface receptor), (4) escape fromendosomes into the cytoplasm by virtue of hydrophobic functional unitsthat integrate into and finally disrupt endosomal membranes, becomingeffective during acidification of endosomes en route to lysosomes, (5)protection by polyethylene glycol (PEG) against degradative enzymeactivities (e.g. peptidases, proteases, etc.).

In this invention a “module” is a biologically active molecularstructure ranging from a small drug molecule or chromophore molecule toa complete protein molecule such as an antibody or lectin. In the caseof the examples presented herein (1) is represented by morpholinoantisense oligonucleotides against α-4 chain and β-1 chain of laminin-8[34, 51] coupled to an intervening spacer by an amide linkage by meansof an —NH₂ (amino) group artificially introduced at their 3′-termini.The spacer is attached to the carrier by a disulfide moiety that iscleavable in the sulfhydryl-disulfide exchange reaction with glutathionin the reducing milieu of the cytoplasm [51, 52]. (2) Tissue targetingis designed by employing a monoclonal antibody (mAB) to recognize andbind rat transferrin receptor. This receptor has been found expressed onendothelium cell surfaces that function as the blood brain barrier(BBB), and at elevated levels on certain tumors [53, 54]. In vitro andin vivo studies indicate that transferrin receptor may be used as ananchorage for a drug delivery system chemically bound to transferrin ormAB OX-26 or any other appropriate mAB that binds the transferrinreceptor and thereby achieves transcytosis through blood brain barrier(BBB) of rat or mouse or other mammals depending of the allotype of theantibody [5, 56, 57; 45, 58, 59, 60, and 61]. (3) Antibody binding totransferrin receptor and internalization into endosomes has beendemonstrated [57, 55, and 57. It will be appreciated that in the case ofthe transferrin receptor any appropriate antibody, mAB, humanized orchimeric antibody or lectin or other ligand specific to the transferrinreceptor can be used. It is also appreciated that appropriate ligands toany number of cell surface receptors or antigens can be used in theinvention and that transferrin receptor is merely an example. (4)Endosomal escape has been shown to function for polyacrylic acidderivatives by acidification during maturation of the endosomal vesiclestowards lysosomes [51, 62]. The designed carrier proposed in this patentapplication carries an abundance of valine residues linked to thepolymalic acid scaffold by amide bonds. During acidification of theendosomes en route to lysosomes, these stretches of the carrier moleculebecome charge-neutralized and hydrophobic, and are capable to disruptmembranes. Other molecules may be used in place of valine so long asthey become charge neutralized at lysomal pH's. (5) PEGylation markedlyincreases the half-life of conjugated proteins [63], prolongs thecirculation time, and enhances extravasation into targeted solid tumors[64]. Any other molecule know to increase half-life may be used in theinvention.

DESCRIPTION OF THE FIGURES

FIG. 1 a shows the overall structure of a drug molecule of the presentinvention.

FIG. 1 b shows the overall sequence of steps used to assemble thestructure of FIG. 1 a.

FIG. 2 is a diagram illustrating the synthesis of PMLA-NHS ester;

FIG. 3 is a diagram illustrating the synthesis of (PDP-morpholino)antisense oligonucleotides;

FIG. 4 is a diagram illustrating the synthesisN-fluorescein-5′-thiocarbamoyl)diaminohexane;

FIG. 5 is a diagram illustrating the synthesisN,N′-bis-(3-maleimidopropionyl)poly(ethylene glycol);

FIG. 6 is a diagram illustrating the synthesis ofPMLA/L-valine/2-mercaptoethylamine/mPEG5000-NH2 conjugate

FIG. 7 is a diagram illustrating the synthesis of PMLA/mABOX-26/morpholino antisenseoligonucleotide/L-valine/2-mercaptoethylamine/mPEG5000-NH2 conjugate

FIG. 8 is a diagram illustrating the conjugation of FITC to PMLA/mABOX-26/morpholino antisenseoligonucleotide/L-valine/2-mercaptoethylamine/mPEG-NH2 conjugate;

FIG. 9 shows a bar graph of the results of a hemolytic assay used intesting the polymers.

FIG. 10 a shows the release of morpholino oligos from the carrier bymeans of reduction with glutathione.

FIG. 10 b shows the percentage of oligo release over time in response tof reduction with glutathione.

FIG. 11 shows a Kaplan-Meir survival curve of rats after treatments withDrug 2 compared to Drug 2A and/or PBS (mock) as analyzed by a log ranktest with significance at p<0.01;

FIG. 12 is Co-distribution of endosomal marker FM 4-64 with Drug 2 (30min) in cultured glioma U-87MG cells. By confocal microscopy, FM 4-64 isseen in the cytoplasmic endosomes (upper left), and Drug 2 is found inthe same place (upper right). Both labels co-localize (lower left,yellow color);

FIG. 13 is a chart illustrating that vessel density is increased intumors compared to normal brain and that Drug 2 reduces tumor vesseldensity by 55%;

FIG. 14 shows immunofluorescence staining of U87MG glioma cultures forlaminin chains. PMLA vehicle-conjugated antisense oligos (Drug 2)inhibit laminin expression (α4 is red and β1 is green). Nuclei arecounterstained with DAPI (blue);

FIG. 15 shows immunofluorescence analysis of xenotransplanted tumorsusing a monoclonal antibody to human laminin β1 chain; laminin β1 chainsynthesis was inhibited in GBM after Drug 2 administration; and

FIG. 16 shows western blot analysis of inhibition of laminin-8 chainexpression in two glioblastoma multiforma (GBM) cell cultures, U87MG andT98G: −, no treatment, +, treatment with Drug 2.

FIG. 17 shows that Drug 2 is capable of crossing the BBB where the redcolor represents the drug visualized within brain vessels andtransplanted tumor cells following intravascular administration of thedrug.

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided to enable any person skilled inthe art to make and use the invention and sets forth the best modescontemplated by the inventor of carrying out his invention. Variousmodifications, however, will remain readily apparent to those skilled inthe art since the general principles of the present invention have beendefined herein specifically to provide a novel drug delivery system asexemplified by an antisense anti-tumor drug based on poly-L-malic acid.

The attractive properties of PMLA as a carrier matrix or moleculartransport vehicle for pharmaceuticals and biopharmaceuticals are thefollowing: it is non-toxic and non-immunogenic; its hydrophobicity canbe controlled by introducing hydrophobic side chains or spacers [30]; itis biodegradable [31]; and it is stable in bloodstream. For targetingantisense oligos to a specific organ or compartment, targeting entitiessuch as tumor-specific antibodies that favor receptor-mediatedendocytosis can be conjugated to the PMLA polymer. Ideally, the systemincludes a releasing system for releasing the drug from the moleculartransport vehicle; possible releasing systems include: a) a disulfidebond cleavable by the intracellular glutathione, b) a pH-sensitivehydrazone bond, c) a tetrapeptide cleaved by lysosomal cathepsin B,which activity is elevated in various tumors (or other pepidases); d) anintrinsic release function from endosome [32, 33]; and e) other labileor cleavable bonds such as ester linkages. Most importantly, inhibitorsof multiple molecular targets can be easily attached to one PMLAmolecule.

PMLA from the natural source, plasmodia of Physarum polycephalum [27 andreferences therein], was the starting material for the synthesis of thedrug delivery vehicle described in the present patent application. Themethods of chemical syntheses employed here are from the general fundusof methods in synthetic chemistry, and have been described in othersystems, not related to the polymalic acid-based system described here.Most of these methods had to be adapted to the present situation, inparticular to the properties of educts during the progress of thechemical construction of the carrier system, and with regard to themethods of purification of products. To achieve a successfulderivatization with a predictable and reproducible stoichiometry of thefunctional moieties conjugated to the polymalic acid scaffold, thesequence of the reactions with the scaffold had to be established andorganized in such a way, that an uncontrolled reaction was impossible.The validity of products has been achieved and the purity controlled byin situ analysis during the stepwise synthesis, including qualitativeand quantitative chemical assays, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), and ultraviolet/visible/IRlight spectroscopic analyses as well as NMR-spectroscopic methods. Themembrane disruption properties of the fully assembled drug vehicle andalso of the intermediates of its synthesis was assessed by a routine,empirical hemolytic membrane assay [see 51].

The references of standard synthesis methods underlying the developmentof the methods used here are in particular:

-   -   Activation of PMLAH-carboxylic groups as N-hydroxysuccinimide        (NHS) esters in analogy to the method of [69].    -   Coupling of the (2-pyridyldithio)propionyl group to morpholino        (PDP)-morpholino) antisense oligonucleotides in analogy to the        method described in [51 and references therein].    -   FITC (fluorescein isothiocyanate)-conjugation in analogy to the        method for the formation of        N-(fluorescein-5′-thiocarbamyl)diaminohexane of [71].

Synthesis of N,N′-bis-(3-maleimidopropionyl)poly(ethylene glycol) inanalogy to the method of [70].

-   -   Introduction of thiol groups into antibodies in analogy to the        techniques described of [72, 51 and references therein].    -   Reaction of mAB OX-26-sulfhydryl with        N,N′-bis-(3-maleimidopropionyl)-PEG diamide conjugate were        performed in analogy to the reactions carried out in [52, and        69].    -   Synthesis of PMLA/L-valine/2-mercaptoethylamine/mPEG-NH2        conjugate by amide formation from the NHS-activated carboxylate        was carried out principally as described herein.    -   Synthesis of PMLA/mAB OX-26/morpholino antisense        oligonucleotide/L-valine/2-mercaptoethylamine/mPEG-NH2 conjugate        by reaction of sulfhydryl group with maleimide;    -   Conjugation of FITC-spacer to PMLA/mAB OX-26/morpholino        antisense oligonucleotide/L-valine/2-mercaptoethylamine/mPEG-NH2        conjugate, represented in principal an amide formation by        nucleophilic attack of the NHS-activated carboxylate as        described herein.

Existing drug delivery systems suffer from one or several of thefollowing problems:

-   -   They are not multifunctional, i.e. they are limited with regard        to variability in the kind and amount of tissue targeting groups        per carrier molecule;    -   They are limited by the kind and number of conjugated drugs        (pro-drugs) per carrier molecule;    -   They are limited by solubility in physiological fluids;    -   They are limited by insufficient stability against degradation        in the circulation system;    -   They are not biodegradable;    -   They involve viral nucleic acids or other viral fragments;    -   They are not specific for tumor tissue and they damage healthy        host tissue    -   They are toxic;    -   The synthesis of the drug delivery system suffers from        uncontrollable side reactions;    -   The synthesis of the drug delivery system suffers from        solubility or other problems that render purification of        reaction products difficult or impossible;    -   The synthesis of the drug delivery system does not result in        reproducible products;    -   The synthesis of structural variations/extension to contain new        components, thus enhancing specificity or sharpening the        antitumor activity of the drug delivery system is not possible;        and    -   The synthesis of the drug delivery system cannot be readily        scaled up.

The controlled conjugation of each reactive functional module with theNHS(N-hydroxysuccinimide) activated carboxylic groups of the polymalicacid scaffold allows one to conjugate a variation of different kinds ofreactive functional modules, thus introducing a variety of differenttargeting molecules, drug (pro-drug) molecules, etc. The various modulescan be conjugated to one and the same scaffold molecule or to differentones allowing a binary or ternary, etc. drug mixture. Multiplefunctional modules on one and the same scaffold molecule can displaybiologically synergistic effects when simultaneously being introducedinto the cell.

Biodegradability can be achieved by employing biodegradable polymalicacid as scaffold and other biodegradable building units (amino acids,proteins).

DESCRIPTION OF THE INVENTION BY A SPECIFIC EXAMPLE

Synthesis of a Polymalic Acid Based Multifunctional Carrier System forthe Tumor Targeted Delivery of Morpholino Antisense Oligonucleotides

FIG. 1 a shows the overall structure of a typical drug molecule of thepresent invention. For the synthesis of Block (z+w), the carboxyl groupsof polymalic acid (block w) are activated as NHS-esters and conjugatedto L-valine via amide bond. Block y3 is the pro-drug morpholinoantisense oligos containing a disulfide drug releasing unit. Block y1 isthe monoclonal antibody (OX26) targeting molecule conjugated to theblock z+w via polyethylene glycol (PEG) spacer. Block x is thePEG-protector against degradation, attached by an amide bond and formedfrom commercially available PEG-amine. Block y2 represents remainingsulfhydryl anchor groups, which have not been consumed by the synthesisat this point, and which can be used for the conjugation of additionalfunctional modules via reaction with the double bond of substitutedN-ethylmaleimides or simply blocked by reaction with unsubstitutedN-ethylamaleimide. Block n, the fluorescent reporter group, is preparedfrom fluorescein isothiocyanate (FITC) and N1-Boc-1,6-diaminohexane. Thedrug structure is built by a stepwise coupling of the blocks (i.e., themodules) onto the growing conjugate as shown in FIG. 1 b under a carefulstoichiometric control. The order of the steps can be readily adjustedto fit different scenarios. Conjugations are carried out withcarbodiimide reagents in organic solvent, preferably dimethylformamide.Side reactions are prevented by appropriate protection of side chainsfollowing standard methods.

For Block w highly purified polymalic acid is obtained from cultures ofPhysarum polycephalum [24, 25]. The biopolymer is spontaneously andenzymatically degraded to L-malic acid [31, 26], which is metabolized tocarbon dioxide and water. The sodium salt is neither toxic norimmunogenic in mice and rabbit respectively [27 and references therein].After intravenous injection into mice, polymalate was rapidly cleared byexcretion via the kidneys [73]. Certain polyymalate derivatives andblock polymers actually showed positive effects on bone repair andmuscle regeneration in rats [30] or were found biocompatible in otherinvestigations [32]. Polymalic acid is an excellent candidate for thedesign of a drug carrier device, because of its high abundance ofmodifiable carboxyl groups. These can be easily conjugated to a varietyof different biologically active molecules in a perfectly controllablefashion regarding their stoichiometry and integrity. Block z is based onpolymers that contain lipophilic groups like L-valine or L-leucine andbecome increasingly lipohilic when protonated when ambient pH fallsbelow pH 6 during maturation of endosomes to lysosomes. This increasinglipophilicity results in leakiness of the endosomal membranes and causesrelease of the macromolecular content into the cytoplasm [33, 74, 75].Block y3 contains a disulfide bond, which is stable in blood circulationincluding brain microvessels but is cleaved in the reductive environmentof the cells [38]. Block y1 contains a polyethylene glycol spacer thatallows the tissue targeting moiety to bind to the receptor on the targetcell surface. It also protects against degradation of the targetingpolypeptide. (y3) The morpholino oligonucleotides, which specificallyblock the expression of tumor essential genes, such as the α1-chain oflaminin are used. In principle, any other drug or pro-drug can beconjugated here, as well as an array of different drugs on a singlecarrier molecule. These conjugates are cleaved from the carrier at thedrug releasing unit within the cytoplasm, and the drug(s) becomeeffective. Block y1 helps breach the BBB which is targeted by amonoclonal antibody against the transferrin receptor on the endothelialcells of the BBB [55]. Bradykinin alone or conjugated together withother molecules, might also be a targeting molecule to be used for thebrain tumors by virtue of specific receptors [77, 18, and 78]. A furtherpossibility for the brain tumor specific targeting is to use amonoclonal antibody against the human EGF receptor [37, 79]. BradykininB₂ receptors and EGFR are overexpressed on tumor cells and can also beused as brain tumor targeting sites in combination with transferrinreceptor. Block n adds an arbitrary fluorescent label, here fluorescein,which is conjugated to the drug structure to facilitate homing studiesof the carrier in the endosomes of recipient tumor cells.

Materials and Methods

Poly(β-L-malic acid) (PMLA) was purified from the broth of culturedPhysarum polycephalum plasmodia using methods developed from [25]. Thepolymer in salt form was size fractionated on Sephadex G25 columns. Thefraction with a number-averaged molecular mass of 50 kDa (polydispersity1.2) was converted to the free polymer acid (PMLA-H) by passage overAmberlite IR-120 (H⁺ form) and stored freeze-dried before used incarrier synthesis. ¹H-NMR in D₂O gave the following δ-values: 3.3 ppm(doublet, the methylene protons of the polyester backbone), 5.3 ppm(triplet, the methine protons of the polyester backbone).Proton-broad-band-decoupled ¹³C-NMR gave the following δ-values: 178.4ppm (—COOH), 74.5 ppm (—CHOH—), 38.9 ppm (—CH₂—), and 174.5 ppm (—CO—).Purified PMLA-H shows UV-light absorbance only below 220 nm wavelength,and is devoid of absorbance at 260 and 280 typical for nucleic acids andproteins, respectively (further details are reviewed in [27]).Morpholino™-3′—NH₂ antisense oligonucleotides [6] to the α-4 chain oflaminin-8 (AGC-TCA-AAG-CCA-TTT-CTC-CGC-TGA-C) and to the β-1 chain oflaminin-8 (CTA-GCA-ACT-GGA-GAA-GCC-CCA-TGC-C) [50, 34] were purchasedfrom Gene Tools (USA). Mouse monoclonal antibody against rat transferrinreceptor CD71 (clone OX-26, isotype IgG_(2a)) at a concentration of 1mg/ml PBS containing 10 mM sodium azide was obtained from ChemiconEurope (UK). Mouse IgG_(2a), κ(UPC 10) was purchased from Sigma(Germany). Chromatographically pure mPEG-amine (MW 5000) andamine-PEG-amine (MW 3400) were obtained from Nektar Therapeutics (USA).Reagents and solvents obtained from Merck (Germany), Sigma (Germany),Pierce (USA) were of the highest available purity. Dichloromethane (DCM)and N,N-dimethylformamide (DMF) were dried over molecular sieves (0.4nm).

¹H-NMR spectra were recorded on a Bruker Model DMX-500 Fourier transformspectrometer and chemical shifts are given in ppm (δ) relative to TMS asinternal standard. ¹³C NMR spectra were recorded on the samespectrometer operated at 125.8 MHz. Chromatographic separations wereperformed with a Merck-Hitachi analytical LaChrom D-7000 HPLC-systemequipped with UV and fluorescence detectors. Either Macherey & NagelC₁₈-Nucleosil reversed-phase (RP) columns (250×4 mm) with a binarygradient of 0.1% TFA (trifluoroacetic acid) in water −0.07% TFA inacetonitrile at a flow rate of 1.5 ml/min or size exclusion columnsBio-Sil SEC 250-5 (5 μm, 300×7.8 mm) with 50 mM sodium phosphate bufferpH 7.4 at a flow rate of 0.75 ml/min were used. Molecular mass of thepolymer Na or K-salt was determined by SEC-HPLC with polystyrenesulfonate standards of defined molecular weight (Machery-Nagel). Thinlayer chromatography (TLC) was performed on Merck precoated silica gel60 F254 aluminum sheets. The eluent contained a mixture of n-butanol,water, and acetic acid (4:2:1 on a volume ratio basis).

Syntheses Synthesis of PMLA-NHS Ester

1.16 g of PMLA-H (10 mmol regarding the malic acid monomer) wasdissolved in 30 ml of anhydrous dimethylformamide (DMF).N-hydroxysuccinimde (NHS) (15 mmol), dissolved in 10 m of anhydrousdimethylformamide (DMF), was added to the PMLA-H solution. Thetemperature was lowered to 0° C. in an ice bath, thendicyclohexylcarbodiimide (DCC) (15 mmol) dissolved in 10 ml of DMF wasadded. The reaction mixture was held under reduced pressure at roomtemperature until no gas bubbles developed. After 30 min at 0° C., thereaction mixture was stirred at room temperature for 48 h. The reactionmixture was held as described above under reduced pressure followed byincubation every 2 h during the first day of reaction, then every 6 hduring the second day of reaction. After two days of reaction,dicyclohexylurea was removed by filtration, and the reaction volume wasreduced by evaporation under reduced pressure. Fresh anhydrous DMF (10ml) was added and residual dicyclohexylurea was again removed byfiltration. The clear reaction mixture was stirred for 12 h at roomtemperature and last amounts of dicyclohexylurea (if any) were removedby filtration. The volume was reduced to 1-3 ml by evaporation underreduced pressure, and the product was precipitated by the addition ofethyl acetate. The pale yellow product (P1) was collected by filtration.Diethylether was added to the filtrate to match the final proportion of1:1 (ethyl acetate:diethylether), and more of a light brown product wascollected by filtration (P2). Then n-hexane was added to the filtrate tomatch the final proportion of 1:1:1 (ethylacetate:diethylether:n-hexane) and additional brown product wascollected by filtration (P3). The precipitates were dispersed in thesame solvents used for their precipitation and left overnight in thecold (−20° C.). The products were filtered and washed repeatedly withthe same cold solvents. The products were further purified by passagethrough Sephadex LH 20 using DMF as eluent allowing the flow by gravity.The product containing fractions were collected and the solventsevaporated under reduced pressure. Finally, the products were dispersedin diethylether, collected by filtration, dried in vacuo, and stored at−20° C.

The purity/composition of these preparations of PMLA-NHS ester wasanalyzed by ¹H NMR and UV-VIS spectroscopy. The content of NHS groupswas determined after aminolysis of NHS ester groups with n-butylamine.10 mg of PMLA-NHS ester were dissolved in 0.5 ml of DMF. A portion of0.5 ml of 10% n-butylamine was added to this solution, and the reactionmixture was incubated at room temperature for 30 min. Aftercentrifugation, samples of 20 μL were mixed with 80 μl of water andanalyzed by RP-HPLC employing water/0.1% (v/v) TFA as eluent. NHS groupswere monitored by their absorbance at 260 nm. Their content wascalculated by comparing the absorbance with that of standards of knownamounts of N-hydroxysuccinimide. The molar ratio of malic acid residuesand NHS-groups in the PMLA-NHS ester sample was calculated by combiningthese results with the amounts of malyl residues measured by ¹H-NMR.Typically, the ratios were 35, 59, and 85% for P1, P2, and P3,respectively. ¹H NMR in (CD₃)₂SO gave the following δ-values: 2.8 ppm(singulet, 4H N—CO—CH2-), 3.35 ppm (doublet, the methylene protons ofthe polyester backbone), 5.85 ppm (triplet, the methine protons of thepolyester backbone). The reaction is shown in FIG. 2.

Synthesis of (2-pyridyldithio)propionyl-morpholino(PDP-morpholino)antisense oligonucleotides

Morpholino-3(—NH₂ antisense oligomer (1 μmol) was dissolved in a of 900μL of DMF and 100 mL of deionized water. To this mixture, 20 μL of a 100mM solution of N-succinimidyl 3×2-pyridyldithio)propionate (SPDP) in DMFwas added and left for 2 h at room temperature. The solvent was removedby rotary evaporation under reduced pressure at room temperature. Theresidue was dissolved in 1 ml of buffer A (0.1 M sodium phosphate, 0.15M NaCl, pH 7.2) containing 10 mM EDTA and purified over a Sephadex G-25microspin column pre-equilibrated with buffer A. The concentration ofPDP-morpholino antisense oligonucleotide was adjusted to 1 mM and storedat −20° C.

The purity of the product was confirmed by TLC and UV-spectroscopy byshowing the absence of NHS and SPDP. The content of PDP groups wasdetermined by measuring the concentration of 2-thiopyridone afterdisulfide reduction as follows: PDP-morpholino antisense oligonucleotidewas incubated with 0.2 M dithiothreitol (DTT) in 0.1 M Tris buffer pH9.0 for 30 min at room temperature. The reaction mixture was subjectedto RP-HPLC by first washing for 10 min with distilled water and theneluting in 30 min with a gradient of 0-60% acetonitrile. The reactionproduct 2-thiopyridone was detected using UV absorption at 341 nm. Theconcentration of 2-thiopyridone was measured by using the absorbance ofknown amounts of reduced 2-aldrithiol (DPDS) as standards. The yield ofPDP-morpholino antisense nucleotide was routinely higher than 80% of thestarting amount of morpholino-3′-NH₂ oligonucleotide. This reaction isshown in FIG. 3.

Synthesis of N-(fluorescein-5′-thiocarbamoyl)diaminohexane

Fluorescein isothiocyanate isomer I (90 mg) (FITC, minimum 98%, 0.23mmol) was dissolved in 3 ml DMF, and 76 mg of N₁—Boc-1,6-diaminohexanehydrochloride (0.3 mmol) were added. The coupling reaction was startedby dropwise addition of 0.6 mmol of triethylamine. The reaction mixturewas incubated for 2 h at room temperature, and the volume was reduced byevaporation under reduced pressure (final volume approximately 0.5 ml).Cold water (5 ml) was added to the remaining mixture and acidified with1 N HCl. The precipitate was collected by centrifugation, washed threetimes with cold water followed by centrifugation, until no trace ofN₁—Boc-1,6-diaminohexane could be detected in the supernatant asdetermined by TLC and ninhydrin test. The final product was dried overP₂O₅. This synthesis is illustrated in FIG. 4.

To remove the Boc protecting group, the dried product was dissolved in 3ml of dichloromethane (DCM), and the temperature was lowered with an icebath. Two ml TFA were added to the solution which was then stirred for30 min on ice. The reaction was followed by TLC. Fluorescent spots werevisible under UV light. The solvent was evaporated under reducedpressure, and the waxy product was dissolved in acetone and precipitatedby the addition of diethylether. For purification, the product wasdissolved in 3 ml of DCM/ethanol (3:2, v/v) containing 4 ml of aceticacid in 100 ml mixture and passed through a 2 cm×12 cm SiO₂ columnequilibrated with the same solvent. The product was pure by TLC. TheRf-values were 0.95 for FITC, 0.98 forN₁-(fluorescein-5′-thiocarbamoyl)-N₆—BOC-1,6-diaminohexane, and 0.64 forN-(fluorescein-5′-thiocarbamoyl)diaminohexane.

Synthesis of N,N′-bis-(3-maleimidopropionyl)poly(ethylene glycol)

0.5 g of NH2-PEG3400-NH₂ (0.147 mmol) dissolved in 3 mL of anhydrous DMFwas added dropwise to (3-maleimidopropionic acid NHS ester) (106 mg, 0.4mmol) dissolved in 5 ml of anhydrous DMF with vigorous stirring at roomtemperature. The completeness of the reaction was confirmed by TLC and anegative ninhydrin test. After incubation for 2 h at room temperature,the solvent was removed by rotary evaporation at room temperature underreduced pressure. The product was dissolved in 2 ml of buffer A (0.1 Msodium phosphate, 0.15 M NaCl, pH 7.2) containing 10 mM EDTA. Insolubleimpurities were removed by centrifugation. The clear supernatant waspassed over a Sephadex G-25 column pre-equilibrated with buffer A. Theproduct was pure by TLC and ninhydrin test. The aqueous solution of theproduct was stored at −20° C.

¹H-NMR spectra of the product dissolved in (CD₃)₂SO indicated thefollowing δ-values: 7.05 singlet 4H—HC═CH—, 3.74 triplet 2H N—CH2, 3.5singlet hydrogens from PEG, 3.03 triplet 2H CH₂—CONH. The values wereconsistent with the product being the expectedN,N′-bis-(3-maleimidopropionyl)poly(ethylene glycol) diamide. Thecontent of maleimido groups was measured indirectly by a method relyingon the reaction of —HC═CH— with the sulfhydryl of given amounts of2-mercaptoethylamine (2-MEA) and the titration of unreacted sulfhydrylwith 5,5′-dithiobis-2-nitrobenzoate (DTNB, Ellman's reagent) as follows.An appropriate amount of 2-MEA in water was added to the aqueoussolution of N,N-bis-3-maleimidopropionyl)poly(ethylene glycol) diamideand incubated for 30 min at room temperature. DTNB (25 μL solution of 10mg/mL in ethanol) was added and the absorbance at 412 nm read after 10min incubation at room temperature. The absorbance was standardized withknown amounts of 2-MEA, and the amount of unreacted sulfhydryl groupscalculated. The content of maleimide groups in the sample ofN,N′-bis-(3-maleimidopropionyl)poly(ethylene glycol) diamide wascalculated by subtraction of the amount of unreacted 2-MEA from theinitial amount of 2-MEA. From this value, the yield of the synthesis ofN,N′-bis-(3-maleimidopropionyl)-poly(ethylene glycol) diamide wascalculated to be 65%. This synthesis is shown in FIG. 5.

Introduction of Thiol Groups into Antibodies: Reduction of IntrinsicDisulfide Bonds with 2-mercaptoethylamine (2-MEA)

Mouse monoclonal antibody (mAB) against rat transferrin receptor CD71(clone OX-26, isotype IgG_(2a)) was commercially obtained at aconcentration of 1 mg/ml PBS containing 10 mM sodium azide. Mousemonoclonal antibody IgG_(2a)κ (UPC 10, Sigma) in place of mAB OX-26 wasused to generate a control conjugate and also a standard curve forprotein measurements. The mAB OX26 solution was concentrated using amicrocentrifuge membrane filter (Sigma Ultrafree-CL microcentrifugefilters, regenerated cellulose, cutoff 30 kDa) at 4° C. and 5000×g. ThemAB storage buffer was changed to buffer A (0.1 M sodium phosphate, 0.15M NaCl, pH 7.2) containing 10 mM EDTA. The concentration of the mAB wasadjusted to 3-5 mg/ml. Mouse mAB UPC 10 was dissolved in buffer A togive the same concentration as mAB OX 26. Solid 2-mercaptoethylaminehydrochloride (2-MEA) (6 mg/ml) was stirred into the antibody solutionsand the mixture incubated for 90 min at 37° C. The disulfide-reducedantibodies were purified by diafiltration with buffer A (degassed underN₂) using microcentrifuge membrane filters (regenerated cellulose, cutoff. 30 kDa) at 4° C. and 5000×g. The step was performed until thediafiltrate was completely free of 2-MEA as measuredspectrophotometrically at 412 nm after incubation with5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent). The numberof thiol groups in the disulfide-reduced antibody solutions wascalculated by reading the absorbance at 412 nm (e=14.15×10³ M⁻¹ cm⁻¹ at25° C.) after 10 min incubation of 0.5 ml antibody solution with 25 μlof DTNB solution (10 mg/ml in ethanol) at pH 8.0. The antibody proteinconcentration was measured according to method of [65]. The resultsindicated that the disulfide-reduced antibodies contained 3.1-3.5 thiolgroups per molecule of IgG-molecule. SEC-HPLC analysis showed only onepeak of MW 150 kDa indicating that the reduced antibody had maintainedits molecular integrity. The preparations were rather stable in thepresence of EDTA even in the absence of reducing agent, and nosignificant losses in free thiol groups were observed overnight at 4° C.

Synthesis of mAB OX-26/N,N′-bis-(3-maleimidopropionyl)-PEG DiamideConjugate

Immediately after preparation, the solution of reduced antibody wasadded dropwise to the stirred aqueous solution ofN,N′-bis-3-maleimidopropionyl)-PEG diamide (in 50-fold molar excess ofmaleimide groups over the thiol groups of the antibody) at roomtemperature. The reaction mixture was incubated for 2 h at roomtemperature, and the unreacted N,N′-bis-(3-maleimidopropionyl)-PEGdiamide was removed by dialysis in buffer A (50 kD membrane, overnightwith three times buffer change). The preparation ofantibody-N,N′-bis-(3-maleimidopropionyl)-PEG diamide conjugate was usedimmediately for further synthetic reactions.

The completeness of the conjugation reaction was indicated by theresults of SEC-HPLC. When measuring absorbance at 220 nm wavelength, theonly material eluted was in the position of 7.20-7.26 min whichrepresented the conjugate, while none eluted in the position of13.4-13.7 min for the unreacted N,N′-bis-3-maleimidopropionyl)-PEGdiamide. The content of maleimide groups in the antibody conjugate wasdetermined indirectly using 2-MEA and Ellman's reagent as describedabove for the synthesis of N,N-bis-(3-maleimidopropionyl)-PEG diamide.The antibody protein concentration was determined according to method of[65]. The results indicated 3.0 maleimido groups per antibody moleculeand were in agreement with the assumption that the free thiol groups ofthe disulfide-reduced antibodies preparations had fully reacted withN,N′-bis-(3-maleimidopropionyl)-PEG diamide. Moreover, because SEC-HPLCanalysis showed only one peak of MW 150 kDa, products of crosslinkingcould be excluded.

Synthesis of PMLA/L-valine/2-mercaptoethylamine/mPEG₅₀₀₀-NH₂ Conjugate

For the synthesis of PMLA/L-valine/2-mercaptoethylamine/mPEG-NH₂conjugate, 1 mmol (with regard to malyl units) of PMLA-NHS ester(preparation P3: 85% NHS ester) was dissolved in 10 ml of anhydrous DMF.First, mPEG5000-NH2 (50 μmol in 2 mL DMF, corresponding to 5 mol-% ofthe NHS activated malyl units) and 200 μmol of N-ethylmorpholine wereadded in this sequence and the mixture stirred at room temperature for30 min until the reaction was completed according to TLC with theninhydrin test (negative versus positive ninhydrin reaction at origin).Next, 200 μmol of a 50 mM solution of 2-MEA in DMF (corresponding to 20mol-% of NHS-activated malyl groups) and 200 μmol of N-ethylmorpholinewere added to the reaction mixture with stirring for 30 min at roomtemperature. Again, the reaction was complete according to TLC (Rf=0.27for 2-MEA and Rf=0 for the polymer conjugate) with the ninhydrinreaction. The synthesis is shown in FIG. 6.

According to the stoichiometry of the added reagents, 4.5% and 19.3% ofthe PMLA-NHS ester equivalents had been replaced by PEG and 2-MEA,respectively. The remaining unreacted NHS esters equivalents wereallowed to conjugate to L-valine (1 mmol in 5 ml water), added dropwisein the presence of 0.1 g of NaHCO₃ (1.2 mmol). The reaction mixture wasstirred for 1 h at room temperature and neutralized under cooling with0.1 M HCl. The solvent was evaporated at 30° C. under reduced pressure.The dried product was dissolved in 10 ml of buffer A (0.1 M sodiumphosphate, 0.15 M NaCl, pH 7.2) containing 10 mM EDTA and 1 ml of 0.5 MDTT. After 10 min at room temperature, the mixture was centrifuged at 20000×g for 5 min and the clear supernatant passed over a Sephadex G25column (2.5 cm×60 cm) pre-equilibrated with buffer A, and the productcontaining fractions lyophilized.

The composition of the PMLA/L-valine/2-mercaptoethylamine/mPEG-NH₂conjugate was analyzed by ¹H NMR and UV-VIS spectroscopy. The content ofthiol groups was measured by the addition of 25 μl of DTNB solution (10mg/ml in ethanol) to 1 mg of lyophilized conjugate dissolved in 1 ml ofsodium phosphate buffer (pH 8.0, 100 mM) and reading the absorbance at412 nm wavelength after 30 min incubation at room temperature. In caseof the preparation of PMLA/L-valine/2-mercaptoethylamine/mPEG-NH2/FITCconjugate (see below), the reaction mixture was also diafiltrated with amicrocentrifuge membrane filter (regenerated cellulose, cut off 5 kDa)at 5000×g before reading the 412 nm absorbance. The Ellman method forassaying thiols is based on the reaction of thiols with the chromogenicDTNB (5,5′-dithiobis-2-nitrobenzoate, FW 396.4) whereby formation of theyellow 5-thio-2-nitrobenzoic acid (TNB) is measured.

The reason for filtration in case of FITC-conjugate is to separate TNBfrom FITC-conjugate because the presence of fluorescence makes thedetection of TNB impossible. During the filtration TNB passes themembrane, FITC-conjugates are retained, and the absorbance of thefiltrate is measured (The filtration is not for removal of free dye. Thefree dye is already removed before this reaction).

The sulfhydryl content was calculated with regard to 2-MEA standards.The content of L-valyl moieties was determined by quantifying the freeamino groups after total hydrolysis using the trinitrobenzenesulfonicacid (TNBS) assay and RP-HPLC as follows: 1 mg of the conjugate and30-50 μL of 6 N HCl were placed in a 100 μL capillary tube. The sealedcapillary was incubated in an oven at 100° C. for 12 to 16 hrs. Afterhydrolysis the contents were transferred into an Eppendorf tube (rinsingthe capillary tube with water to be quantitative) and evaporated tocomplete dryness by gentle warming. This material was redissolved inwater and centrifuged. A 10-30 μl aliquot of the supernatant was addedto 300 μl of sodium bicarbonate buffer (0.4 g NaHCO₃ in 10 ml water, pH8.5). After addition of 150 μL of 0.1% (w/v) TNBS aqueous solution, themixture was incubated for 30 min at 37° C. After centrifugation, 20 μLof the reaction mixture were separated by RP-HPLC using a lineargradient of 30 min (0-10 min, 100% water, 10-40 min from 0-60%acetonitrile). The content of valyl moieties was calculated on the basisof the 340 nm absorbance in the eluent with regard to known amounts ofL-valine as standards. The molar ratio of mPEG:valine: 2-MEA of theconjugate was determined by ¹H NMR.

Synthesis of PMLA/mAB OX-26/morpholino antisenseoligonucleotide/L-valine/2-mercaptoethylamine/mPEG-NH₂ Conjugate

Freshly prepared mAB OX-26-PEG-maleimide was added dropwise toPMLA/L-valine/2-mercaptoethylamine/mPEG-NH₂ conjugate at 4° C. withstirring. A 100 molar excess of free thiol groups (conjugate) overmaleimido groups was applied, allowing all antibodies to becomeconjugated with the polymer. After 30 nm in of incubation, the reactionwas complete. The completeness of the reaction was confirmed bySEC-HPLC. The PMLA/mAB OX-26/L-valine/2-mercaptoethylamine/mPEG-NH₂conjugate was purified by diafiltration in buffer A usingmicrocentrifuge membrane filters (regenerated cellulose, MW cut off of100 kDa) at 4° C. and 5000×g. The mAb-containing PMLA conjugate wasretained by the filter and only protein-freePMLA/L-valine/2-mercaptoethylamine/mPEG-NH₂ conjugate passed through thefilter. The diafiltration was repeated until no trace of theprotein-free polymer conjugate was detected in the diafiltrate asconfirmed with SEC-HPLC.

The protein content of the PMLA/mABOX-26/L-valine/2-mercaptoethylamine/mPEG-NH₂ conjugate was measured bythe method of [66]. The same amount of unconjugated mAb showed anapproximately 10% higher absorbance, and thus, the measured proteincontent was corrected by factor of 0.9. (This factor was empiricallyderived from the fact that, although no antibody was found to leakthrough the diafiltration, the retained (conjugated) antibody amountedto only 90% of the educt antibody.) The reason for this discrepancy isnot known. The concentration of the remaining free thiol groups of theprotein-containing conjugate was determined as described above. It wasfound that approximately 70% of the initial thiol groups were stillpresent. The concentration of the protein-containing conjugate wasadjusted to 3 mM with regard to thiol groups.

In the next step, 5 μmol of PDP-morpholino antisense oligonucleotidesfor α4 and β1 chains of laminin 8 (5 ml of the 1 mM solution ofPDP-morpholino antisense oligonucleotides) was added dropwise withstirring to the purified solution of the PMLA/mABOX-26/L-valine/2-mercaptoethylamine/mPEG-NH2 conjugate equaling aconcentration of 15 μmol of free thiol groups (5 ml of 3 mM solution ofthe protein-containing conjugate). The molar ratio of antisense α4 chainto the antisense β1 chain was 1:1. The reaction mixture was incubatedovernight at 4° C. The completeness of the reaction was confirmed bySEC-HPLC indicating a single peak in the eluent with 260 nm absorbanceand the absence of absorbance in the positions of free PDP-morpholinoantisense oligonucleotides. The obtained PMLA/mAB OX-26/morpholinoantisense oligonucleotide/L-valine/2-mercaptoethylamine/mPEG-NH₂conjugate was purified by diafiltration using microcentrifuge membranefilter (regenerated cellulose, cut off: 100 kDa) at 4° C. and 5000×gcentrifugation. The content of the free sulfhydryl groups at this stagewas determined with Ellman's reagent at 412 nm. The content of antisensemorpholino oligonucleotides was measured by absorbance at 260 nm afterreduction of the disulfide groups with 50 mM DTT for 2 h at 37° C. andseparation by SEC-HPLC. Specifically, the 260 nm light absorbing peakswere compared with those obtained for reduced PDP-morpholino antisenseoligonucleotides as standards.

The protein content of the PMLA/mAB OX-26/morpholino antisenseoligonucleotide/L-valine/2-mercaptoethylamine/mPEG-NH2 conjugate wasmeasured by the method of [65]. The molar ratio of antibody: morpholinoantisense oligonucleotide varied from 1:20 to 1:26 resulting a y1-valueof 0.19%-0.25% (see FIG. 7 for overall synthesis). Free sulfhydrylgroups were blocked with N-ethylmaleimide, and unreacted reagent wasremoved by diafiltration as above.

Conjugation of FITC to PMLA/mAB OX-26/morpholino antisenseoligonucleotide/L-valine/2-mercaptoethylamine/mPEG-NH2 Conjugate

For detection under biological conditions, PMLA/mAB OX-26/morpholinoantisense oligonucleotide/L-valine/2-mercaptoethylamine/mPEG-NH2conjugate was covalently labeled with fluorecein isothiocyanate (FITC).This label was, however, introduced after conjugation of mPEG-NH₂ toPMLA-NHS esters indicated above. The solution ofN-(fluorescein-5′-thiocarbamoyl)diamino-hexane was prepared in a mixtureof DMF and PBS (1:1) to a final concentration of 25 mM. To the solutionof mPEG-NH₂/PMLA conjugate (1 mmol with regard to malyl units ofPMLA-NHS ester, preparation P3: 85% NHS ester) 25 μmol ofN-fluorescein-5′-thiocarbamoyl)diamino-hexane (n=2.5% in FIG. 7) and 100μmol of N-ethylmorpholine were added, and the mixture was incubated atroom temperature for 30 min. The reaction was followed by TLC. Aftercompletion of the reaction, fluorescence was detected only at theorigin. The construction of the FITC to PMLA/mAB OX-26/morpholinoantisense oligonucleotide/L-valine/2-mercaptoethylamine/mPEG-NH₂conjugate then followed the same route as described above in the absenceof the dye. The content of FITC in the conjugates was measured byabsorption of the FITC-moiety at 490 nm. FITC-carrying conjugates werealso detected by a Merek-Hitach fluorescence detector with theexcitation wavelength set to 447 nm and the emission wavelength set to514 nm. The content of FITC was calculated by comparing the absorbanceor fluorescence of samples with that of standard samples generated byquantitative conjugation of varying amount of FITC to carrier polymer asdescribed above. The conjugate was used directly for cell cultureexperiments. This synthesis is shown in FIG. 8.

Hemolysis Assay. FIG. 9 shows the membrane disruptive activity of thepolymers as measured using a red blood cell (RBC) hemolysis assay. Thefresh human RBCs were isolated by centrifugation of whole human blood at2000 g for 5 min. The RBCs were washed three times with cold 100 mMsodium phosphate buffer of the desired pH (pH 5.85 or pH 5.5). The finalpellet was resuspended in the same buffer to give a solution with 108RBCs per 1 ml. The polymers were dissolved in 100 mM dibasic sodiumphosphate buffer at the desired pH at a concentration of 10 mg/ml. Thepolymer concentration was 2.5 nmol/10⁸ RBCs. Hemolysis in distilledwater was used to produce 100% lysis. RBCs in buffer with no polymer wasused as reference controls. The hemolysis assay was performed by addingthe polymer solution to the suspended RBCs in 1 ml of the appropriatebuffer. The RBCs were mixed by inverting the tube several times, andincubated for 1 h in a 37° C. water bath. After incubation, the RBCswere centrifuged for 10 nm in at 13,500×g to sediment intact cells andthe lysis was determined by measuring the absorbance of the supernatantsat 541 nm which reflects the amount of hemoglobin released by the RBCs.The relative increase 100(A-A_(o))/A_(total) in absorbance at 541 nmwavelength of the cell-free supernatant was measured as an indicator ofmembrane rupture. The results show that PMLA-PEG-Valine,PMLA-PEG-Valine-AS, and PMLA-PEG-Valine-AS-mAB infer membranedestabilization in contrast to PMLA-PEG which stabilizes RBC membranes.Comparison shows that destabilization is due to the presence ofPMLA-conjugated valine. At decreasing pH (simulating maturation ofendosomes to become lysosomes), the carboxyl groups of valine becomeprotonated, and destabilization increases due to an increasedlipophilicity of the charge-neutralized valine moieties.

Release of the Morpholino Oligos. FIGS. 10 a and 10 b shows the releaseof morpholino antisense oligonucleotides from the drug carrier due tothe cleavage of the disulfide bond by glutathion (glutathion-SH). Thecleavage of the disulfide bond is a two-step reaction. In the firststep, one equivalent of glutathion-SH reacts with the disulfide forminga mixed disulfide antisense oligonucleotide —S—S-glutathion (stripedcolumns) (FIG. 10 a) and one equivalent of free antisenseoligonucleotide-SH (solid columns). Over time the mixed disulfidedecreases and the free antisense increases. This reaction is rapid as isseen in FIG. 10 b. In the second step, the mixed disulfide reacts with asecond equivalent of glutathion to yield the disulfideglutathion-S—S-glutathion and free antisense oligonucleotide-SH. Thissecond reaction is slow. The two-step mechanism and the relative ratesof the reactions are typical for this so called disulfide exchangereaction. The results show that the oligonucleotides are veryefficiently cleaved from the drug carrier in the cytoplasm, whichcontains glutathion at this given concentration.

To mimic cytoplasmic release of antisense morpholino oligonucleotidesfrom the drug vehicle as shown in the figures, 5 mM GSH(γ-L-glutamyl-L-cysteinylglycine, MW 307.33) was added to the reactionmixture at room temperature. The mixture contained 0.25 mM of the drugvehicle in water. At various times, the reaction was stopped by theaddition of an excess of N-ethylmaleimide (20 mM final concentration)over total sulfhydryl moieties. The reduced antisense was detected asN-ethylmaleimidyl antisense. The reaction products were separated byHPLC and the released antisense morpholinos were detected by their UVabsorbance at 260 nm. The results are shown in FIG. 10 b. A Complete(100%) release is referenced to the reduction in the presence of 50 mMDTT at 37° C. for 1 h. HPLC analysis was performed with a Merck-Hitachanalytical HPLC unit using a gel filtration column. Separation wascarried out on a (300×7.7 mm) Macherey & Nagel 125-5 GFC-HPLC columnusing sodium phosphate buffer (50 mM, pH 7.4) with a flow rate of 0.75ml/min.

Treatment of Human Glioblastoma Grown in Brain of Nude Rat withLaminin-8 Antisense Oligonucleotides Conjugated to Poly-L-Malic Acid

Specific drug delivery is crucial for treating tumors and reducing sideeffects for normal cells. Simultaneous inhibition of several moleculartargets at the level of protein synthesis may be highly effective inpreventing tumor growth and progression. Laminin-8 chains overexpressionis associated with glioma progression, and laminin-8 blocking inhibitsglioma invasion in vitro [34].

Methods

Polymalic acid (PMLA). A multifunctional drug delivery constructconsists of modules attached to the pendant carboxyl groups of polymalicacid (PMLA). The polymer is a natural product of Physarum polycephalum[27]. The modules are (1) morpholino antisense oligonucleotidesconjugated to the scaffold by disulfide bonds, which bonds are cleavedin the cytoplasm to release the free drug, (2) antibodies againsttransferrin receptor for cancer cell targeting and receptor-mediatedendocytosis, (3) short chain PEG-conjugated L-leucine and directlycoupled L-valine, both linked through amide bonds, to providepH-dependent lipophilicity to disrupt endosomal membranes, (4) longchain PEG for increasing time in circulation, and (5) fluorescentreporter molecules (fluorescein, Cy5 or similar fluorophores) to detectthe construct molecule within the tissue/cell.

Scheme of Drugs used for the animal treatment.

-   -   Drug 1: antisense oligo to laminin α4+ antisense oligo to        laminin β1 (α4:β1=1:1);    -   Drug 2: antisense oligo to laminin α4+ antisense oligo to        laminin β1 (α4:β1=1:1)+monoclonal anti-transferrin receptor        antibody (antibody OX-26 to rat CD71 from Chemicon        International) as vehicle for delivery to the fast dividing        cells [35, 36, 37, and 38];    -   Drug 3: antisense oligo to laminin α4+ antisense oligo to        laminin β1+antisense oligo to EGFR (α4:β1:EGFR=1:1:1);    -   Drug 4: antisense oligo to laminin α4+ antisense oligo to        laminin β1+ antisense oligo to EGFR        (α4:β1:EGFR=1:1:1)+anti-transferrin receptor antibody.    -   Drugs 1A, 2A, 3A and 4A were identical to the drugs of the        corresponding number (i.e., Drug 1 was identical to Drug 1A)        except that the corresponding sense oligos were used in place of        the antisense oligos.

Drug 1: antisense oligo nucleotide to laminin α4 chain and antisenseoligo nucleotide to laminin β1 chain conjugated to PMLA; Drug 2:antisense oligo nucleotide to laminin α4, antisense oligo nucleotide tolaminin β1, and monoclonal anti-transferrin receptor antibody (antibodyOX-26 from Chemicon International) conjugated to PMLA. Controls (Drugs1A and 2A) were the same carrier conjugates with the antisense oligosreplaced by corresponding sense oligos. The human U-87MG glioblastomacell line was used for in vitro experiments and injected intracraniallyinto NIHRNU-M NIH nude outbred homozygous rats (Taconic Inc.).

NIH Nude outbred rats (Tac:N:NIH-Whn, Taconic) were used for thesetests. For antisense treatment, we used Morpholino oligos (Gene Tools,LLC) as the most specific, stable and effective both in vitro and invivo. Morpholinos have been used in the past successfully for in vitrostudies, but in vivo, their delivery has been less successful [6,7].Poly-L-malic acid (PMLA) was used as a delivery carrier for getting theMorpholinos into the cells. The principle of the method for purificationof PMLA has been described [25]. A modern scaled-up PMLA productionmethod [27] was used. The chemistry of PMLA functional groups has beeninvestigated [28, 29], showing that the chemical derivatization andpurification of the products in both organic and aqueous solvents isreadily achievable. For our experiments, PMLA was chemically conjugatedto a monoclonal antibody against transferrin receptor, to make the drugmost specific for fast dividing cells [35, 36, 37, and 38], in additionto targeting tumor-specific laminin-8 chains.

Preliminary toxicity studies were performed for morpholinooligonucleotides, PMLA and their conjugate after complete synthesis. 30days after injection of each chemical, gross and micro-pathologicalanalysis were performed, and no abnormal changes were noted. Animals didnot develop neurological abnormalities and their appetites were alsonormal. Animal test subjects were created by injecting humanglioblastoma U-87MG cells intracranially using a stereotactic device.The drug treatments started three days after the injection of tumorcells.

For intracranial treatment, rats were injected with antisense oligos ondays 3, 7, 10 and 14 (four treatments total) as diagrammed below.

Groups of 12 rats each were injected with oligos to laminin-8 α4+β1chains at doses of 0.5 mg/kg or 2.5 mg/kg. Control groups of 11 ratseach were injected with sense oligos to α4 chain+β1 chain at 0.5 mg/kgor at 2.5 mg/kg. All surgical and non-surgical procedures were performedaccording IACUC protocol 001118, dated August of 2003. For intracarotidtreatment, a group of rats had a catheter implanted into the carotidartery right after the tumor implantation. The catheter was connected toan implantable subcutaneous injection port. The rats were given infusionof 900 μl of antisense and sense oligos solution (0.06 ml per minute for15 minutes with a peristaltic pump) into the right carotid artery viathe subcutaneous port chamber followed by heparin flush. Rats wereeuthanized in 30 minutes after the end of infusion. The controlconsisted of: (a) 3 rats that were euthanized on day without any kind oftreatment to obtain normal control tissue, and (b) 4 rats with tumorsthat were sham-injected intracranially with PBS on days 1, 3, 7, 10 and14, and euthanized for tissue harvest as soon as they developedneurological symptoms caused by tumor progression.

Results

Intracranial tumor treatment Drug 2 doses of 0.5 and 2.5 mg/kg wereequal for the treatment in the survival study. After intracranialadministration of four doses of Drug 2, the animal survival time wasincreased by 30%, p<0.008 (FIG. 11), compared to rats treated with PBS(mock) or sense oligos (Drug 2A). Two treatments, however, only produceda marginal effect. Drug 1 without transferrin receptor antibody did notaffect survival. Therefore, the mechanism of drug cell delivery isprobably transferrin receptor-mediated endocytosis. Interestingly,addition of antisense to EGFR to Drug 2 (Drug 4) resulted in loss ofactivity, possibly due to increased glioma cell survival in hypoxicconditions with EGFR inhibited [39].

The mechanism of drug internalization was investigated in culturedglioma cells. When cells were treated with fluorescein-labeled Drug 2and rhodamine-labeled endosomal marker FM 4-64 (Molecular Probes,Eugene, Oreg.) the staining for both compounds showed co-localization.In 10 minutes, stains co-localized near cell membrane and in 30 minutesboth labels were found in the endosomes (FIG. 12).

If cells were pretreated with transferrin receptor antibody and thentreated with Drug 2 in ten minutes, the drug was not seen in thecytoplasm (data not shown). These results suggest that transferrinreceptor antibody is required as part of the active drug because itallows drug penetration into cells by receptor-mediated endocytosis,after which the antisense oligos can be released within the targetcells. If the cells are pretreated with free antibody to transferrin,the receptors are blocked and the antibody on the drug is unable tobind.

We found that Drug 2 decreases vessel density and specific laminin chainexpression in human gliomas xenotransplanted to nude rats. Wedemonstrated that Drug 2 designed to inhibit laminin-8 expressionreduces vessel density in tumors. These vessels were visualized byimmunostaining for von Willebrand factor. The number of vessels wascounted in both drug-treated and untreated animals, in five microscopicfields on three serial sections (15 fields per tumor) under ×200magnification, using a Zeiss Axioscop microscope connected to an imagecapturing system (Hamamatsu, Japan). The data were input into NIH ImageJsoftware to quantify the vessels. Statistical significance wasdetermined by ANOVA.

Angiogenesis. As shown on FIG. 13, microvascular density in the U87MGhuman tumors without treatment was significantly higher than in normalbrain. After four intracranial treatments with Drug 2 tumor vesseldensity was reduced by 55%. Data are presented for control brains ofthree sham-operated (normal) rats (45 microscopic fields), five rats (75microscopic fields) with untreated tumors, and five rats with Drug2-treated tumors (75 microscopic fields). The results confirm theantiangiogenic mechanism of action of Drug 2 designed to inhibitlaminin-8 expression.

Laminin chain immunostaining. It was important to show that Drug 2 infact inhibited the expression of targeted laminin-8 chains. To this end,experiments were conducted both in vivo and in vitro. For tumorimmunostaining, an antibody to human laminin-8 β1 chain was used thatdoes not recognize rat laminin but reacts with tumor-derived laminin.FIG. 14 shows the effect in cell culture where the antisense effectivelyeliminates the immunostaining. As shown in FIG. 15, Drug 2 alsoeffectively reduced immunostaining for laminin β1 chain inxenotransplanted human tumors.

Inhibition of laminin-8 expression in vitro was assessed in conditionedmedia of two cultured human gliomas, U87MG and T98G, treated for 3 dayswith Drug 2 Western blot analysis on FIG. 16 shows marked reduction oflaminin α4 chain (three-fold decrease by densitometry) and disappearanceof laminin β1 chain. Therefore, Drug 2 efficiently inhibits theexpression of target laminin chains both in vivo and in vitro.

Intracarotid and Intravenous Tumor Treatment In Vivo

Fourteen days after human U87-MG glioma cells inoculation into rat brainfor intracarotid treatment, Group of 3 rats had a catheter implantedinto the carotid artery right after the tumor implantation. The catheterwas connected to an implantable subcutaneous injection port. The ratswere given infusion of 900 pt of antisense and sense oligos solution(0.06 ml per minute for 15 minutes with a peristaltic pump) into theright carotid artery via the subcutaneous port chamber followed byheparin flush. Drug 2 was injected at a concentration of 2.5 μg/kg forintracarotid treatment or via the tail vein at a concentration of 5μg/kg (3 rats as well). The drug distribution was examined at 1, 3, 12and 24 hours after injection. Using the fluorescence unit, we detectedthe drug in transplanted tumor cells (heavy staining) and vascular cells(lighter staining) of the brain (FIG. 17). Drug 2 was visualized bymeans of a rhodamine stained antibody that labels (in red) thetransferrin antibody carried by Drug 2. The cell nuclei arecounterstained with DAPI (blue). Rat brain (right panel) shows limited,mainly vascular, staining while the transplanted U87MG tumor cells (leftpanel) are heavily labeled. Maximum concentration in these locations wasachieved in 3 and 12 hours time-point after drug injection. Theseresults confirm that Drug 2 penetrates the blood-brain barrier (BBB),possibly by means of receptor-mediated endocytosis.

Conclusions. An in vivo model has been developed that is suitable forstudying laminin-8 expression and its inhibition in human tumors. Thecombination of antisense oligos to laminin-8 α4 and βchains (blockage oflaminin-8) combined with a novel drug delivery vehicle, PMLA,efficiently inhibited laminin-8 expression in a xenografted intracranialhuman glioma in rats. After a preliminary four-times antisensetreatment, the survival of treated animals with glioma was significantlyincreased, with p<0.008. These data indicate that PMLA-based antisensedrugs using laminin-8 as a therapeutic target are effective ininhibiting human brain tumors.

The following claims are thus to be understood to include what isspecifically illustrated and described above, what is conceptuallyequivalent; what can be obviously substituted and also what essentiallyincorporates the essential idea of the invention. Those skilled in theart will appreciate that various adaptations and modifications of thejust described preferred embodiment can be configured without departingfrom the scope of the invention. The illustrated embodiment has been setforth only for the purposes of example and that should not be taken aslimiting the invention. Therefore, it is to be understood that; withinthe scope of the appended claims, the invention may be practiced otherthan as specifically described herein.

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1. A drug delivery molecule comprising: a polymerized carboxylic acidmolecular scaffold having a plurality of free carboxylic acid groups; aplurality of biologically active molecular modules, each beingcovalently linked to the same polymerized carboxylic acid molecularscaffold, wherein said active modules comprise: at least one targetingmodule for promoting cellular uptake by a target cell; and at least onepro-drug module for altering cellular metabolism of the target cell. 2.The drug delivery molecule according to claim 1, wherein the pro-drug isselected to inhibit expression of tumor-specific proteins.
 3. The drugdelivery molecule according to claim 1, wherein the polymerizedcarboxylic acid molecular scaffold is poly(β-L-malic acid).
 4. The drugdelivery molecule according to claim 3, wherein the poly(β-L-malic acid)has a molecular mass between 2,500 and 100,000.
 5. The drug deliverymolecule according to claim 4, wherein the poly(β-L-malic acid) has amolecular mass of at least about 5,000.
 6. The drug delivery moleculeaccording to claim 1, wherein each molecule of the polymerizedcarboxylic acid molecular scaffold has at least about 50 free carboxylicacid groups.
 7. The drug delivery molecule according to claim 1, whereinthe plurality of molecular modules further includes a molecular modulefor promoting disruption of biomembranes.
 8. The drug delivery moleculeaccording to claim 7, wherein said molecular module for promotingdisruption of biomembranes comprises a molecule having lipophiliccharacteristics and groups that are charged at physiologic pH and becomeuncharged at lysosomal pH thereby increasing lipophilicity of saidmolecular module.
 9. The drug delivery molecule according to claim 1,wherein the plurality of active molecular modules further includes amolecular module for prolonging circulation of the drug deliverymolecule.
 10. The drug delivery molecule according to claim 9, whereinthe molecular module for prolonging circulation of the drug deliverymolecule comprises polyethylene glycol.
 11. The drug delivery moleculeaccording to claim 1, wherein the plurality of active molecular modulesfurther includes a reporter module for determining cellular uptake ofthe drug delivery molecule.
 12. The drug delivery molecule according toclaim 11, wherein the reporter module comprises a fluorescent molecule.13. The drug delivery molecule according to claim 1, wherein thetargeting molecule is selected to promote penetration of the blood brainbarrier.
 14. The drug delivery molecule according to claim 1, whereinthe targeting molecular module comprises an antibody.
 15. The drugdelivery molecule according to claim 14, wherein the antibody binds to atransferrin receptor.
 16. The drug delivery molecule according to claim14, wherein the antibody is a monoclonal antibody.
 17. The drug deliverymolecule according to claim 14, wherein the antibody is a humanized orchimeric antibody.
 18. The drug delivery molecule according to claim 1,wherein the pro-drug molecular module is linked to the polymerizedcarboxylic acid molecular scaffold by a cleavable linkage that iscleaved when the drug delivery molecule enters a cell.
 19. The drugdelivery molecule according to claim 18, wherein the cleavable linkageis a disulfide linkage.
 20. The drug delivery molecule according toclaim 1, wherein the pro-drug molecular module comprises an antisensemolecule.
 21. The drug delivery molecule according to claim 20, whereinthe antisense molecule is a morpholino antisense molecule.
 22. The drugdelivery molecule according to claim 20, wherein the antisense moleculeinterferes with production of laminin-8.
 23. The drug delivery moleculeaccording to claim 22, wherein the antisense molecule interferes withproduction of laminin-8 by altering production of a laminin subunitselected from the group consisting of α4 laminin and β1 laminin.
 24. Amethod of synthesizing a drug delivery molecule comprising the steps of:providing a polymerized carboxylic acid molecular scaffold having aplurality of free carboxylic acid groups; activating the carboxylgroups; reacting the activated carboxyl groups with a compoundcontaining sulfhydryl groups and amino groups to add sulfhydryl groupsto the drug delivery molecule to make a sulfhydryl-drug deliverymolecule; reacting a targeting molecule containing a sulfhydryl bindinggroup with the sulfhydryl-drug delivery molecule to promote uptake by atarget cell; and reacting a pro-drug molecule for altering cellularmetabolism of the target cell.
 25. The method of synthesizing a drugdelivery molecule of claim 24, wherein the pro-drug molecule is anantisense molecule containing a sulfhydryl binding group.
 26. The methodof synthesizing a drug delivery molecule of claim 24, further comprisinga step of reacting the activated carboxyl groups with a molecule with alipophilic portion and containing charged groups which become unchargedduring acidification of endodomes thereby causing membrane disruption.27. The method of synthesizing a drug delivery molecule of claim 24,wherein a plurality of different pro-drug molecules are linked to thesame drug delivery molecule, thereby allowing simultaneous treatment ofthe target cell with more than one pro-drug molecule.
 28. The method ofsynthesizing a drug delivery molecule of claim 24, wherein the targetingmolecule is selected to promote penetration of the blood brain barrier.